Spatial modulation of light to determine object position

ABSTRACT

Approaches for determining object position in a flow path are disclosed. A system includes a spatial filter having a length disposed along a longitudinal axis of the flow path and a width along a lateral axis of the flow path. The spatial filter has mask features configured to modulate light. Light emanating from objects moving along the flow path is detected. The detected light has a component along a detection axis that makes a non-zero angle with respect to the longitudinal and lateral axes. An electrical output signal that includes information about the trajectory depth of the object is generated in response to the detected light.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under contract numberW911NF-10-1-0479 (3711), awarded by the Department of Defense. The U.S.Government has certain rights in this invention.

TECHNICAL FIELD

This application relates generally to techniques for performing sampleanalysis by evaluating light emanating from the objects in a sample. Theapplication also relates to components, devices, systems, and methodspertaining to such techniques.

BACKGROUND

The present disclosure relates generally to techniques that determineobject characteristics using light emanating from the objects. Morespecifically, the techniques can use spatial filter and/or maskarrangements to allow for the transmission, reflection, fluorescence,phosphorescence, photoluminescence, chemoluminescence and/or scatteringof light with time variation, such as where the objects are movingrelative to the spatial filter and/or mask arrangements.

Various techniques have been proposed for using light emanating fromobjects. For example, U.S. Pat. No. 7,358,476 (Kiesel et al.) describesa fluidic structure with a channel along which is a series of sensingcomponents to obtain information about objects traveling within thechannel, such as droplets, cells, viruses, microorganisms,microparticles, nanoparticles, or other objects carried by fluid. Asensing component includes a set of cells that photosense a range ofphoton energies that emanate from objects. A processor can receiveinformation about objects from the sensing components and use it toobtain spectral information. Additional techniques are described, forexample, in U.S. Patent Application Publications 2008/0181827 (Bassleret al.) and 2008/0183418 (Bassler et al.) and in U.S. Pat. No. 7,701,580(Bassler et al.), U.S. Pat. No. 7,894,068 (Bassler et al.), U.S. Pat.No. 7,547,904 (Schmidt et al.), U.S. Pat. No. 8,373,860 (Kiesel et al.),U.S. Pat. No. 7,420,677 (Schmidt et al.), and U.S. Pat. No. 7,386,199(Schmidt et al.).

SUMMARY

An assembly includes at least one spatial filter having a length along alongitudinal axis of a flow path and a width along a lateral axis of theflow path. The spatial filter has mask features disposed at leastpartially along the length of the spatial filter and extending at leastpartially across the width of the spatial filter. The mask featuresinclude at least first mask features having a first optical transmissioncharacteristic and second mask features having a second transmissioncharacteristic different from the first transmission characteristic. Atleast one detector is positioned to detect light with respect to adetection axis, where the detection axis makes a non-zero angle withrespect to the longitudinal and lateral axes, in order to determine thetrajectory of the object in the flow path. The detected light is lightemanating from at least one object and time modulated according to themask features as the object moves along the longitudinal axis. Thedetector is configured to generate a time-varying electrical signal inresponse to the detected light that includes information about the depthof the object in the flow path. The system also includes an analyzerconfigured to determine a trajectory depth in the flow path of theobject along the detection axis based on the detector signal.

Another embodiment involves a system that determines object position inthree dimensional space. A first spatial filter is arranged in an x-yplane of a flow path in three dimensional space characterized by x, y,and z axes, the first mask having a first group of mask features. Asecond spatial filter is arranged in an x-z plane of the space, thesecond mask having a second group of mask features. A first detector ispositioned to detect light emanating from at least one object and timemodulated according to the first group of mask features as the objectmoves along the flow path. The first detector generates a firsttime-varying electrical signal in response to the detected lightmodulated according to the first group of mask features. A seconddetector is positioned to detect light emanating from at least oneobject and time modulated according to the second group of mask featuresas the object moves along the flow path, the second detector configuredto generate a second time-varying electrical signal in response to thedetected light modulated according to the first group of mask features.An analyzer is configured to determine positions of the object along thex, y, and z axes based on the first and second signals.

Some embodiments involve a method for determining object position in aflow path using a spatial filter having a length disposed along alongitudinal axis of the flow path and a width along a lateral axis ofthe flow path. The spatial filter has mask features configured to timemodulate light. Light emanating from objects moving along the flow pathis detected. The detected light has a component along a detection axisthat makes a non-zero angle with respect to the longitudinal and lateralaxes. An electrical output signal is generated in response to thedetected light. A trajectory depth in the flow path of the object alongthe detection axis is determined based on the output signal.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present disclosure. The figures and thedetailed description below more particularly exemplify illustrativeembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Throughout the specification reference is made to the appended drawingswherein:

FIG. 1 is an example embodiment of an assembly with a spatial filter,detector, and analyzer configured to determine object characteristicsbased on spatially modulated light;

FIG. 2A is a more detailed schematic view of another example embodimentof a fluidic device, spatial filter, and detector;

FIG. 2B is a schematic view of yet another example embodiment of fluidicdevice, spatial filter, and detector with an optical componentpositioned between the spatial filter and the detector;

FIG. 3 is a schematic view of another example embodiment of an assemblywith an optical imaging element positioned between the object anddetector and the spatial filter positioned adjacent the detector;

FIG. 4 is a schematic view of another example embodiment of an assemblywith the optical imaging element positioned between the light source andthe detector and the spatial filter positioned adjacent the lightsource;

FIGS. 5A through 5I illustrate approaches for determining depth ofobjects moving in a flow path in accordance with various embodiments

FIG. 6A is a perspective view of a spatial filter having mask featurespatterned according to an example embodiment;

FIG. 6B shows the spatial filter of FIG. 6A arranged in relation to alight source, a detection region of a flow path, and a detector;

FIG. 6C is an enlargement of the spatial filter of FIG. 6A;

FIG. 6D is a plot of output signals that result from light modulated bythe spatial filter of FIG. 6A;

FIGS. 6E and 6F, respectively, are top and side views of the spatialfilter of FIG. 6A disposed over a flow path containing two lightemanating objects;

FIG. 6G illustrates plots of the time-varying electrical signalsgenerated in response to the modulated light from each of the twoobjects moving close to/away from the spatial filter of FIG. 5D;

FIG. 6H illustrates plots of the Fourier amplitudes of the electricalsignals from FIG. 6G converted to the frequency domain;

FIG. 7 illustrates I₂/I₃ Fourier amplitude peaks measured on alogarithmic scale plotted against velocity and depth for variousexperiments;

FIG. 8A is a schematic view of a portion of a fluidic device, spatialfilter, and detector with objects having cones of light emanating fromthem;

FIG. 8B is a plot of a modulation envelope that results from the passageof one of the objects of FIG. 8A with cones of light α₂ through the flowchannel past detector;

FIG. 8C is a plot of a modulation envelope that results from the passageof one of the objects of FIG. 8A with cones of light α₁ through the flowchannel past detector;

FIG. 9A is a plan view of another spatial filter disposed in the x-yplane and having mask features patterned according to another exampleembodiment to allow for determination of a lateral position and depthposition of an object within the flow path;

FIG. 9B is a plan view of another spatial filter disposed in the x-yplane and having mask features patterned according to another exampleembodiment to allow for determination of a lateral position and depthposition of an object within the flow path;

FIG. 10 is a plan view of another spatial filter disposed in the x-yplane and having mask features patterned according to another exampleembodiment to allow for determination of a lateral position and depthposition of an object within the flow path;

FIGS. 11A and 11B illustrate spatial filters that include first andsecond features wherein the first features are used to determine lateralposition and the second features are used to determine depth position ofan object;

FIG. 12 is a perspective view of another spatial filter disposed in thex-y plane and having mask features patterned as transmissive trianglesaccording to an example embodiment to allow for determination of alateral position of an object within the flow path;

FIG. 13A is a plan view of the spatial filter of FIG. 12;

FIG. 13B is a plot that shows electrical signals that result from thethree flow trajectories across the filter of FIG. 12;

FIG. 14 is a perspective view of another spatial filter disposed in thex-y plane and having mask features patterned as interdigitatedtransmissive triangles according to yet another example embodiment toallow for determination of a lateral position of an object within theflow channel;

FIG. 15A is a plan view of the spatial filter of FIG. 14; and

FIG. 15B is a plot that shows electrical signals that result from thetwo flow trajectories across the filter of FIG. 14;

FIG. 16 shows a perspective view of a portion of a fluidic device andtwo spatial filters in accordance with some embodiments; and

FIG. 17 is a flow diagram of a method of determining object positionaccording to an example embodiment.

The figures are not necessarily to scale. Like numbers used in thefigures refer to like components. However, it will be understood thatthe use of a number to refer to a component in a given figure is notintended to limit the component in another figure labeled with the samenumber.

DETAILED DESCRIPTION

Various techniques have been proposed for using light emanating fromobjects. These techniques have been functionalized for variousapplications and are generally effective for recognizing and obtainingobject characteristics such as size, charge, porosity, surfacecharacteristics, elasticity, and material composition for particularanalytes. Light emanating from an object can originate from a multitudeof physical processes include: Fluorescence, scattering, up-conversion,second harmonic generation, multi-photon excited fluorescence, Ramanscattering, phosphorescence, absorption etc.

The embodiments described herein can be used to perform a positionand/or movement analysis of an object. The approaches described hereincan be used to determine the object velocity, object position, depth inthe flow channel, and/or object trajectory, e.g., three dimensionalposition and/or trajectory, of moving objects. In some cases, the sizesof the objects is known a priori or can be discerned by the system, andthe system may be calibrated to provide an absolute measurement of theobject position, velocity and depth trajectory in the flow path, thatis, the precise location of the object in the flow channel, at any giventime, and over time. In other cases, where the sizes of the objects areunknown, the system may provide a relative depth between objectstraveling along the flow path.

The determination of object position and/or trajectory is based onspatially modulated light emanating from the object. The embodimentsdescribed herein involve techniques for determining depth along adetection axis, multidimensional position and/or trajectory of objectsin one or more dimensions as the objects travel along a flow path. Thus,the techniques disclosed can be used to determine a depth position ofthe object and/or a depth trajectory referenced to a detection axis, alateral position of the object and/or a lateral trajectory referenced toa lateral axis, and/or a longitudinal position of the object referencedto a longitudinal axis. Determining the trajectory of the object as theobject travels in time along the flow channel can include determiningdirectional vectors of travel in one or more dimension. The trajectorycan be obtained by sampling the object position over time anddetermining trajectory vectors based on the multiple positionmeasurements. Object velocity and/or other object characteristics canalso be obtained.

The embodiments described herein involve the use of at least one spatialmask that can be deployed in a variety of applications, includinganalysis of system properties and/or detection of variouscharacteristics of an analyte in a sample. As the object moves along aflow direction, the object emanates spatially modulated light that isdetected by a detector. The detector generates a time varying outputsignal in response to the sensed spatially modulated light emanatingfrom the object. In some implementations, a non-imaging or non-pixilatedphotodetector can be used to generate a time varying electrical outputsignal based on the spatially modulated light. The use of a non-imagingphotodetector, e.g., a non-pixelated detector, may enhance compatibilitywith high-throughput cytometry. For example, approaches disclosed hereinmay obtain object characteristics such as position, trajectory, etc.,directly with a non-pixelated detector and a suitable spatial maskrather than recording an image sequence for each object, requiringextraction of object information from the images to determine objectcharacteristics.

It will be understood that the techniques, apparatuses, systems, andmethods described herein are applicable to detect various objects suchas analytes or particles present in a sample. The term “object” refersbroadly to any object of interest to be detected. In some applications,objects of interest are particles or analytes that are relatively small,and may be microscopic in size. A given particle or analyte may be orinclude one or a collection of biological cell(s), virus(es),molecule(s), certain proteins or protein chains, DNA or RNA fragments,sub-molecular complex(es), droplets (e.g. oil in water), gas bubbles,microparticles, nanoparticles, beads or other small particles that canbind and carry specific chemicals or other analytes, emulsions, any suchtype of object in an array such as an array of sample wells, and adistinguishable region of a surface such as a small area of a sheet ofpaper or other image-bearing medium; a distinguishable region, could,for example, be a colored spot, or other bit(s) of matter, for example.

In some embodiments, sensors can obtain information about objects byreceiving signals from them; for example, signals in the form of lightcan emanate from an object, whether through emission (e.g. radiation,fluorescence, incandescence, chemoluminescence, bioluminescence, otherforms of luminescence, etc.), scattering (e.g. reflection, deflection,diffraction, refraction, etc.), or transmission, and can be sensed by aphotodetector. Cells or other particles may be treated, e.g., stained ortagged with a suitable fluorescent probe or other agent, in such a waythat they emit light or absorb light in a predictable fashion whenilluminated with excitation light. In this regard, the light emitted bya given excited particle may be fluorescent in nature, or it mayconstitute a form of scattered light such as in the case of elastic orinelastic (Raman scattering) scattered light. For simplicity, the lightthat emanates from (by e.g., scattering, emission, or transmission) anobject is referred to herein as “emanating light” or “light emanating.”It will be understood that the techniques, assemblies, apparatuses,systems, and methods described herein are applicable to detecting allforms of light emanating from an object or constituent parts thereof.

In some embodiments, the detector generates at least one time varyingsignal in response to the spatially modulated light. The time varyingsignal includes information about the object's movement and position. Insome embodiments, the time varying signal can be analyzed in the timedomain to extract the movement and position information and/or to obtainother characteristics of the object. For example, the time varyingsignal may be compared or correlated to a known template signal and/orthe time varying signal may be analyzed by examining variousmorphological characteristics of the time varying signal.

In some embodiments, the time varying signal may be transformed from thetime domain to the frequency domain and the analysis may be carried outon the frequency domain signal. For example, in some cases the Fouriertransform of the time varying signal or portions of the time varyingsignal is used to evaluate the signal. A Fourier transform relates asignal to a set of coefficients of sinusoidal base functions. Thecoefficients are sometimes referred to the amplitude of the Fouriertransform. In some implementations, the absolute value of thecoefficients, or other functions of the coefficients, are referred to asthe amplitude of the Fourier transform. For finite length signals orfunctions one practical Fourier transform is the so called fast Fouriertransform (FFT).

FIG. 1 is an example of an assembly 100 configured to determine objectcharacteristics based on spatially modulated light. The assembly 100includes a light source 112, a mask, e.g., a spatial filter 126, a flowpath, e.g., fluidic device 120, a detector 130, a signal processor 140,and an analyzer 150. Components of the assembly are arranged in acoordinate system that includes a longitudinal axis, designated as thex-axis herein, a lateral axis, designated as the y-axis, and a depthaxis, designated as the z-axis. In the description below, the flowdirection of the flow path and/or flow channel is selected to lie alongthe longitudinal axis of the coordinate system, and the longitudinal,lateral, and depth axes are orthogonal to one another. Those skilled inthe art will appreciate that any coordinate system could alternativelybe selected, the arrangement of the assembly with respect to thecoordinate system is arbitrary and does not change the operation of theassembly, and that non-orthogonal axis systems could alternatively beused.

The device 120 is adapted to receive a sample of interest to beanalyzed. The sample may enter the device 120 at an inlet 121 a thereofand exit the device 120 at an outlet 121 b thereof, flowing generallyalong the x-direction through a flow channel 123 formed betweenconfining members 122, 124. The members 122, 124 may be or compriseplates or sheets of glass, plastic, or other suitable materials. One orboth of members 122, 124 may be a microscope slide or a microscope coverglass, or portion thereof. The members 122, 124 need not, however, beplanar in shape. For example, they may be portions of a unitary tube orpipe having a cross section that is circular, rectangular, or anothershape. Other non-planar shapes are also contemplated. In some cases,confinement of the sample may not be necessary, whereupon one or both ofmembers 122, 124 may be omitted. At least a portion of the confiningmember 122 is transmissive to excitation light emitted by the lightsource 112 at least in an excitation region 123 a. In that regard, lightsource 112 may emit excitation light 112 a towards the fluidic device120.

In some cases, for example, the light source 112 may comprise a laser orlaser diode, a conventional light emitting diode (LED) source or aresonant cavity LED (RC-LED) source. If desired, the light source mayincorporate one or more filters to narrow or otherwise tailor thespectrum of the resultant output light. Whichever type of light sourceis selected, the spectral makeup or composition of the excitation lightemitted by the source 112 is preferably tailored to excite, scatter, orotherwise cause emanation of light from at least some of the objectsthat may be present in the sample, as discussed further below.

The sample is depicted as containing objects 105 that emanate light 107in all directions (only some directions are illustrated). The objects105 may have a variety of characteristics, some of which can bedetermined by the analyzer 150 based on the emanating light 107.

The detector 130 receives time varying light and generates an electricalsignal in response to the time varying light. The time variation in thelight detected by the detector 130 may be the result of interactionbetween the excitation light and an input spatial filter to createspatially patterned excitation light that illuminates the object 105.Alternatively, the time variation in the light detected by the detector130 may be the result of interaction between light emanating from theobjects 105 and an output spatial filter. In some embodiments, thedetector includes an optical filter arranged between the detector andthe objects. An optical filter can be particularly useful when theemanating light is fluorescent light and the optical filter isconfigured to substantially block the wavelengths of the excitationlight and to substantially pass the wavelengths of the light emanatingfrom the objects.

The assembly 100 of FIG. 1 includes the spatial filter 126 (sometimesreferred to as a mask) which can be positioned in various locations.Dashed arrows 126 a and 126 b indicate possible locations of the spatialfilter 126 to provide spatially modulated light and/or modulatedexcitation light. In some configurations, indicated by arrow 126 a, thespatial filter 126 can be arranged disposed between the flow channel 123and the detector 130. In this position, the spatial filter 126 isreferred to as an output spatial mask. In other configurations,indicated by arrow 126 b, the spatial filter 126 can be arranged placedbetween the light source 112 and the flow channel 123. In this position,the spatial filter 126 is referred to as an input spatial filter. Aninput spatial filter may be adapted to transmit light emitted by thelight source by varying amounts along the excitation region 123 a of theflow channel 123. In this configuration, the input spatial filtercreates patterned excitation light in the excitation region 123 a of theflow channel 123. According to various implementations, an input spatialfilter may comprise a physical mask including a sequence or pattern offirst regions that have a first optical transmission characteristic,e.g., are more light transmissive, and second regions that have a secondoptical transmission characteristic, different from the firstcharacteristic, e.g., are less light transmissive. Note that the terms“first” and “second” are used herein to identify different features,groups, characteristics, etc. of various components discussed herein.These terms are used for identification purposes only and are not meantimply any particular order or any particular spatial relationship unlessspecifically indicated. For example, “first” mask features may bearranged upstream or downstream of “second” mask features along the flowdirection.

The input spatial filter may alternatively or additionally comprisemicro-optics or a patterned light source configured to create theexcitation pattern. The excitation pattern can be imaged and/or directedonto the excitation region 123 a using optical components for theimaging (e.g., lenses) and/or direction, (e.g., fiber optics orwaveguides).

In some embodiments an output spatial filter may be utilized andarranged disposed between the objects 105 and the detector 130 at adetection region 123 b of the flow channel. In some embodiments, theexcitation region 123 a and the detection region 123 b overlap. In otherembodiments, there may be partial overlap between the excitation anddetection regions or the excitation and detection regions may benon-overlapping or multiple detection regions and/or excitation regionsmay be used with various overlapping and/or non-overlappingarrangements. In the assembly 100 shown in FIG. 1, the output spatialfilter may be adapted to interact with the light 107 emanating from theobjects 105 in the flow channel 123. In some embodiments, the outputspatial filter may be a physical mask comprising a sequence or patternof first regions that are more light transmissive and second regionsthat are less light transmissive. In some embodiments, color spatialfilters may be used such that a first region of the color spatial filteris more transmissive to a first wavelength band and less transmissive toa second wavelength band and a second region of the color spatial filteris less transmissive to the first wavelength band and is moretransmissive to the second wavelength band.

According to some embodiments of an assembly 100 that include an inputspatial filter, as an object 105 travels in the flow direction 123 c inthe excitation region 123 a of the flow channel 123, light emanatingfrom the light source 112 is alternately substantially transmitted tothe object 105 and substantially blocked or partially blocked fromreaching the object 105 as the object 105 travels along the flowdirection 123 c. The alternate transmission and non-transmission (orreduced transmission) of the excitation light 112 a along the flowdirection 123 c produces time-varying light 107 emanating from theobject 105. The time-varying light 107 emanating from the object 105falls on the detector 130 and, in response, the detector 130 generates atime-varying detector output signal 134.

According to some embodiments of the assembly 100 that include theoutput spatial filter configuration, light 112 a from the light source112 illuminates the object 105, causing the object 105 to emanate light107. As the object 105 travels in the flow direction 123 c in thedetection region 123 b of the flow channel 123, the output spatialfilter alternatively entirely or substantially blocks the light 107emanating from the object 105 from reaching the detector 130 andsubstantially transmits the light 107 emanating from the object 105 tothe detector 130. The alternate substantial transmission and blocking(or partial blocking) of the light 107 emanating from the object 105 asthe object 105 flows through the detection region 123 b produces timevarying light that falls on the detector 130. In response, the detector130 generates the time-varying detector output signal 134.

In some embodiments such as the embodiment of FIG. 1, the analyzer 150may include a signal transform processor 140 that converts thetime-varying detector output signal 134 to a frequency domain outputsignal 136 so as to provide spectral power as a function of frequency.The signal transform processor 140 is shown as part of the analyzer 150in this embodiment, but may be part of the detector in some embodimentsor may comprise separate circuitry in other embodiments. For example, insome embodiments, the signal transform processor may be part of theanalyzer circuitry along with the detector.

For conversion, the signal processor 140 may use known techniques suchas discrete Fourier transform including, for example, a Fast FourierTransform “FFT” algorithm. Thus, the frequency domain output signal 136represents the frequency component magnitude of the time-varyingdetector output signal 134, where the frequency component magnitude isthe amount of a given frequency component that is present in thetime-varying detector output signal 134 or function. The Fourier signalpower is a relevant parameter or measure because it corresponds to thefunction or value one would obtain by calculating in a straightforwardmanner the Fourier transform (e.g. using a Fast Fourier Transform “FFT”algorithm) of the time-varying signal 134. However, other methods ortechniques of representing the frequency component magnitude, or othermeasures of the frequency component magnitude, may also be used.Examples may include e.g. the square root of the Fourier signal power,or the signal strength (e.g. as measured in voltage or current) obtainedfrom a filter that receives as input the time-varying detector outputsignal 134.

In FIG. 1, the time-varying detector output signal 134 and/or thefrequency domain detector output signal 136 can be passed to theanalysis circuitry 151 of the analyzer 150. The analysis circuitry 151is configured to receive the time-varying detector output signal 134and/or the frequency domain detector output signal 136 and to determineone or more spatial characteristics of the object 105 including atrajectory depth within the flow channel 123 of the object 105 basedupon the time-varying detector output signal 134 and/or the frequencydomain detector output signal 136. As will be discussed subsequently,the various embodiments discussed herein provide examples of techniquesfor determining the one or more spatial characteristics of the object105 using various mask designs and processing techniques. As usedherein, the trajectory depth within the flow channel 123 of the object105 is a distance of the object 105 within the flow channel 123 asmeasured along the z-direction of the Cartesian coordinate system ofFIG. 1. Thus, the trajectory depth within the flow channel is a distancegenerally perpendicular to the flow direction 123 c along the flowchannel 123. In some embodiments, the depth can be measured relative toa component such as the filter or one of the confining members.

FIG. 2A and is an enlarged schematic view of a portion of an assembly200 according to another example embodiment. The portion of the assembly200 illustrated in FIG. 2A includes a flow path, e.g., fluidic device220, a detector 230, and a spatial filter 226. The device 220 is adaptedto receive a sample of interest to be analyzed. The sample may enter thedevice 220 at an inlet 221 a thereof and exit the device 220 at anoutlet 221 b thereof, flowing generally in a flow direction 223 c alongthe x-direction through a flow channel 223 formed between confiningmembers 222, 224. As illustrated in FIG. 2A, the one or more objects 205that comprise the sample can have differing trajectory depths d₁, d₂,and d₃ within the flow channel 223 as measured in the z-direction of theCartesian coordinate system illustrated. For convenience, depths d₁, d₂,and d₃ are shown as measured relative to confining member 224, however,such is not always the case, and a different reference point for thedepth measurement could be used. The objects 205 of the sample may havedifferent positions along the flow channel 223 in the x-direction(generally along the flow direction 223 c of the flow channel 223) aswell as different lateral positions in the y-direction of the Cartesiancoordinate system within the flow channel 223.

As discussed previously, the spatial filter 226 may comprise, forexample, a spatial mask. As will be discussed in greater detailsubsequently, the spatial filter 226 may have a plurality of maskfeatures 270. The mask features 270 include regions 270 a having a firstoptical transmission characteristic, e.g., more light transmissiveregions, and regions 270 b having a second optical transmissioncharacteristic, e.g., less light transmissive regions. For simplicity ofexplanation, many examples provided herein refer to mask featurescomprising more light transmissive regions and mask features or regionscomprising less light transmissive regions. However, it will beappreciated that the optical transmission characteristics of the firstand second types of mask features may differ optically in any way, e.g.,the first features may comprise regions having a first opticalwavelength pass band and the second features may comprise regions havinga second optical wavelength pass band different from the first opticalwavelength pass band. The pattern or sequence of first features 270 aand second features 270 b define a transmission function that changesbased on a three dimensional position of a light 207 emanating object205 within the flow channel 223 (i.e., as measured along thex-direction, y-direction, and z-direction of the Cartesian coordinatesystem). This transmission function may be substantially periodic, or itmay instead be substantially non-periodic. The transmission function issensed by the detector 230, which is configured to output thetime-varying output signal discussed in FIG. 1 in response.

In the embodiment of FIG. 2A, the spatial filter 226 may besubstantially monochromatic or polychromatic as desired. In amonochromatic mask, the first mask features 270 a may be more lighttransmissive and may all have substantially the same transmissioncharacteristic, and the second mask features 270 b may be lesstransmissive than the first mask features or may be non-transmissive(opaque) and also all have substantially the same transmissioncharacteristic (different from that of the first mask features 270 a).In a simple case, the transmissive regions 270 a may all be completelyclear, as in the case of an aperture, and the less transmissive regions270 b may be completely opaque, as in the case of a layer of black inkor other absorptive, reflective, or scattering material. Alternatively,the transmissive regions 270 a may all have a given color or filtercharacteristic, e.g., high transmission for light emanating from anexcited object, but low transmission for excitation light.Alternatively, the less transmissive regions 270 b may have a low butnon-zero light transmission, as in the case of a grey ink or coating, ora partial absorber or reflector.

In the embodiment of FIG. 2A, the spatial filter 226 is positionedbetween the objects 205 and the detector 230. In some configurations,the spatial filter 226 may be positioned within the flow channel, suchas along the top surface of confining member 224 as shown in FIG. 2A.The light emanating 207 from the objects 205 interacts with the spatialfilter 226 to provide time modulation of the sensed light that falls onthe detector 230 as the object 205 travels in the flow channel 223. Inthe illustrated embodiment, the spatial filter 226 is positioned betweenthe objects 205 and the detector 230 without additional opticalstructures between the spatial filter 226 and the detector 230.

FIG. 2B is an enlarged schematic view of a portion of an assembly 250according to another example embodiment. In FIG. 2B, the fluidic device220, spatial filter 226, and detector 230 may be the same as thosedepicted in FIG. 2A. The assembly portion 250 shown in FIG. 2B includesan optical element 251 positioned between the spatial filter 226 and thedetector 230. The imaging element 251 may be configured to focus thespatially modulated light onto the detector 230, for example

FIG. 3 is a schematic view of another embodiment of a portion of anassembly 300 according to another example that includes a remote sensingmask. The portion of the assembly 300 illustrated includes a lightsource 312, a spatial filter 326, a flow path, e.g., fluidic device 320,and a detector 330. Similar to the embodiments of FIGS. 1, 2A, and 2B,the device 320 includes an inlet 321 a, an outlet 321 b, a flow channel323 having a flow direction 323 c, and confining members 322, 324. Thespatial filter 326 includes mask features 370 with light transmissiveregions 370 a and less transmissive regions 370 b. In FIG. 3, thespatial filter 326 is positioned between the objects 305 and thedetector 330 and is positioned remotely from the flow channel 323immediately adjacent the detector 330. An optical imaging element 380such as a lens is positioned between the objects 305 and the filter 326and is configured to image light from the objects 305 onto at least oneof the spatial filter 326 and detector 330. The light emanating from theobjects 305 and imaged by the element 380 interacts with the spatialfilter 326 to provide time modulation of the sensed light received bythe detector 330.

FIG. 4 is a schematic view of yet another embodiment of a portion of anassembly 400. The portion of the assembly 400 illustrated includes alight source 412, a spatial filter 426, a flow path, e.g., fluidicdevice 420, and a detector 430. Similar to the previously discussedembodiments, the device 420 includes an inlet 421 a, an outlet 421 b, aflow channel 423 having a flow direction 423 a and confining members422, 424. The spatial filter 426 includes mask features 470 with lighttransmissive regions 470 a and less transmissive regions 470 b. In FIG.4, the spatial filter 426 is positioned between the light source 412 andthe fluidic device 420 containing the objects 405. As shown, the spatialfilter 426 is positioned remotely from the flow channel 423 immediatelyadjacent the light source 412. Interaction between the output light fromthe light source 412 and the spatial filter 426 causes spatiallymodulated excitation light 412 a. An optical imaging element 480 ispositioned between the filter 426 and the objects 405 and is configuredto image the spatially modulated excitation light 412 a onto anexcitation region of the flow channel 423. Additionally, the opticalimaging element 480 may incorporate one or more filters to narrow orotherwise tailor the spectrum of the resultant spatially modulatedexcitation light. The spatially modulated excitation light causes light407 emanating from the objects 405 to be spatially modulated as well.The spatially modulated light emanating from the objects 405 is sensedby the detector 430.

FIGS. 5A through 5H illustrate approaches for determining trajectorydepth of objects moving in a flow path in accordance with variousembodiments. FIG. 5A is a side view of a portion of an assembly 500including spatial filter 526 arranged in a three dimensional spacereferenced to a coordinate system of lateral, longitudinal, and depthaxes. The axes x, y, z used for reference in the illustrated embodimentare orthogonal, but non-orthogonal coordinate axes could alternativelybe used.

FIG. 5B shows a top view of spatial filter 526. The assembly 500includes at least one spatial filter 526 having a length, L, disposedalong the longitudinal axis, x, of a flow path detection region 523 band a width, W, along a lateral axis, y, of the flow path detectionregion 523 b. The spatial filter 526 includes mask features 526 a, 526 bdisposed in a pattern that extends at least partially along the lengththe spatial filter 526 and extending at least partially across the widthof the spatial filter 526. In this embodiment, mask features 526 a maybe referred to as less transmissive (i.e., opaque) and mask features 526b may be referred to as more transmissive (i.e., clear).

For example, the pattern along the length of the spatial filter may be arepeating pattern, a periodic pattern, a random pattern or any otherpattern. The assembly 500 in FIG. 5A includes at least one detector 530positioned to detect light emanating from the objects 501, 502, 503. Thedetected light has a component along a detection axis, z-axis, thatmakes a non-zero angle with respect to the longitudinal and lateralaxes. The detected light is time modulated according to the maskfeatures as the objects move along the flow path 523 in the flowdirection 523 a. The detector 530 is configured to generate atime-varying electrical signal in response to the detected light thatincludes information about characteristics of the objects, such as thedepth position and/or depth trajectory of the objects in the flow path523. The assembly 500 may include an analyzer 540 configured todetermine a depth of the object in the detection region along thedetection axis based on the time varying signal generated by thedetector 530.

For example, as objects 501, 502, 503 move along a flow path withindetector region 523 b, the particles emanate light in all directions.The light emanating from the objects 501, 502, 503 interacts with aspatial filter 526. The light emerges from the spatial filter 526through the clear features 526 b 1, 526 b 2, 526 b 3 in cones of light501 e, 502 e, 503 e and the light is blocked by the spatial filter 526by opaque features 526 a 1, 526 a 2, 526 a 3, and 526 a 4. As shown inFIG. 5A, object 501 is traveling in flow channel at a trajectory depthd1, object 502 is traveling at trajectory depth d2, and object 503 istraveling at trajectory depth d3. Light emanating from object 501emerges from the spatial filter 526, having passed through clear maskfeature 526 b 1 in cone 501 e, which has a cone angle of θe1. Lightemanating from object 502 emerges from the spatial filter 526 at clearmask feature 526 b 2 in cone 502 e, which has a cone angle of θe2. Lightemanating from object 503 emerges from the spatial filter 526 at maskfeature 526 b 3 in cone 503 e, which has a cone angle of θe3.

Light emanating from object 501 at an angle greater than or equal to θe1but less than or equal to blocking angle θb1 is blocked by the spatialfilter 526 by opaque features 526 a 1 and 526 a 2. Some light emanatingfrom object 501 at an angle greater than θb1 can emerge from clearfeatures 526 b 2, 526 b 3 and light emanating from object 501 is alsoblocked by opaque features 526 a 3, 526 a 4.

Light emanating from object 502 within cone 502 e having cone angle θe2passes through clear feature 526 b 2. Light emanating from object 502 atan angle greater than or equal to θe2 but less than or equal to θb2 isblocked by the spatial filter 526 opaque features 526 a 2 and 526 a 3.Some light emanating from object 502 at an angle greater than θb2 canemerge from clear features 526 b 1, 526 b 3, however, light emanatingfrom object 502 is also blocked by opaque features 526 a 1, 526 a 4.

Light emanating from object 503 within cone 503 e having cone angle θe3passes through clear feature 526 b 3. Light emanating from object 503 atan angle greater than or equal to θe3 but less than or equal to θb3 isblocked by the spatial filter 526 by opaque features 526 a 3 and 526 a4. Some light emanating from object 503 at an angle greater than θb3 canemerge from clear features 526 b 1, 526 b 2, however, light emanatingfrom object 503 is also blocked by opaque features 526 a 1, 526 a 2. Thecone angles θe1, θe2, θe3 of the cones 501 e, 502 e, 503 e are inverselyrelated to the depth of the objects, and θe1>θe2>θe3 for d1<d2<d3. Thecone angles θb1, θb2, θb3 of the blocking cones 501 b, 502 b, 503 b areinversely related to the depth of the objects, and θb1>θb2>θb3 ford1<d2<d3.

FIGS. 5C and 5D illustrate an object 505 moving through along flow path523 at a trajectory depth d5 along the detection axis, z. FIG. 5Cillustrates the object 505 at the moment when it is passing over clearfeature 526 b 2 of the spatial filter 526. Object 505 emanates light 515a that passes predominantly through clear feature 526 b 2 and also somelight 515 b emanating from object 505 passes through clear features 526b 1 and 526 b 3. Due to the proximity of object 505 to the spatialfilter 526, very little or no light passes through clear feature 526 b4. FIG. 5D illustrates the object 505 at the moment when it is passingover opaque feature 526 a 3 of the spatial filter 526. Object 505emanates light 516 a that passes through clear features 526 b 2, 526 b3. Due to the proximity of object 505 to the spatial filter 526, verylittle light 516 b from object 505 passes through clear features 526 b1, 526 b 4.

FIG. 5E is a graph of a portion of an idealized time modulated detectorsignal 531 generated by the movement of object 505 relative to thespatial filter 526. The amplitude of the signal 531 increases anddecreases as the object 505 passes over clear and opaque features,respectively. The peak regions include maximum peaks 531 a of the signal531 that have a maximum value of Max1 due to the amount of lightemanating from the object 505 that passes through the clear features asthe object 505 passes over the clear features as shown in FIG. 5C. Thetrough regions include minimum peaks 531 b of the signal 531 have anoffset of Min1 because of the emanating light that passes through clearfeatures as the object 505 passes over the opaque features asillustrated by FIG. 5D.

FIGS. 5F and 5G illustrate an object 506 moving through along flow path523 at a trajectory depth d6 with respect to the detection axis, z. FIG.5F illustrates the object 506 when it is passing over clear feature 526b 2 of the spatial filter 526. Light 517 b emanating from the object 506passes through clear feature 526 b 2. Some light 517 a, 517 c fromobject 506 passes through clear feature 526 b 1 and 526 b 3. Due to thedistance between object 506 and the spatial filter 526, some light 517 dpasses through clear feature 526 b 4.

FIG. 5G illustrates the object 506 when it is passing over opaquefeature 526 a 3 of the spatial filter 526. Object 506 emanates light 518b, 518 c that passes through clear features 526 b 2, 526 b 3 and alsosome light 518 a, 518 d from object 506 passes through clear features526 b 1 and 526 b 4.

FIG. 5H is a graph of a portion of an idealized time modulated detectorsignal 532 generated by the movement of object 506 relative to thespatial filter 526. The amplitude of the signal 532 increases anddecreases as the object passes over clear and opaque features,respectively. The peaks 532 a of the signal 532 have a maximum value ofMax2 due to the amount of light emanating from the object 506 thatpasses through the clear features as the object 506 passes over a clearfeature as shown in FIG. 5G. The valleys 532 b of the signal 532 have anoffset of Min2 because of the emanating light that passes through clearfeatures as the object 506 passes over an opaque feature as shown inFIG. 5H. For d6>d5, Max2<Max1 and Min2>Min1. The differences in the peakmaxima and minima occur due to the blocking and emitting cones thatresult from the interaction of the emanating light with the mask. Thepeak maxima and peak minima of the time varying signal can be analyzedto determine the trajectory depth in the flow channel of the objectalong the detection axis of the flow path as indicated by FIGS. 5E and5H and as discussed in more detail below. The discussion in connectionwith FIGS. 5A through 5H presumes a uniform light source illuminatingthe objects. In addition, the discussion presumes that light exitingfrom the detection area 523 b through the clear features 526 b does notexperience any change in index of refraction. In many embodiments, theillumination light is non-uniform over the detection area 523 b whichsuperimposes a similar non-uniformity on the time varying output signalfrom the detector. Differences in refractive index between the materialsdisposed on either side of the clear features 526 b of the mask changethe angle of the light exiting the mask according to Snell's law, sinθ₁/sin θ₂=n₁/n₁, where n₁ is the index of refraction of the materialwithin the flow path detection region 523 b, θ ₁ is the angle of lightbefore the light exits through the clear features of the mask 526, n₂ isthe index of refraction of the material between the flow path materialand the detector, and θ₂ is the angle of the light after it exitsthrough the mask.

Furthermore, consider the pulses generated by light emanating from anobject that is traveling very close to the mask features such that thetrajectory depth is small when referenced from the position of the mask.The resulting signal, illustrated as signal 592 of FIG. 5I, will becomecloser to a square wave, instead of the sinusoidal shape shown in FIG.5E. Because of the geometry resulting from the close proximity of theobject to the mask features when the object is close to the spatialfilter, emanating light predominantly reaches the detector through oneclear opening directly below the object. The angles are such that lightgoing through other mask openings are at such shallow angles, much ofthe light will be directed away by the opaque features and fail to reachthe detector. When the object is close to the mask, the emanating lighteither reaches the detector when the particle is over a clear feature,or is blocked when the particle is over an opaque feature, resulting ina predominantly square wave shape as illustrated by signal 592. Theactual wave shape may be affected by the object length relative to themask feature length as described and illustrated in concurrently filedU.S. patent application Ser. No. 14/181,530, which is incorporated byreference in its entirety.

When the object is farther from the mask, emanating light from theobject reaches the detector through a clear opening directly below theobject and a portion of the light reaches the detector through otherclear features. Because the object is farther from the mask features,the emanating light generates in a detector signal 591 that includespulses having a longer rise time, t_(r1), when compared to the risetime, t_(r2), of pulses of the signal 592 generated when the object iscloser to the mask features. As the object moves farther away from themask features, the pulses more and more resemble a sine wave and areless and less square wave shaped. Thus, the shape of the signalgenerated by the emanating light, e.g. rise times and/or fall times ofthe pulses, can be analyzed in addition or alternatively to the peakminima and maxima to determine the trajectory depth in the channel.

In some embodiments, the spatial filter selected for use may enhancesignal processing for depth analysis. FIG. 6A illustrates a spatialfilter 626 with a pattern of features that produces an output signalhaving two frequency components. Features of the two frequencycomponents can be analyzed in the time domain, or can be converted tothe frequency domain, e.g., using a Fourier transform, for analysis.

FIG. 6A shows a perspective view of a portion of a fluidic device 620and spatial filter 626. The fluidic device 620 includes a flow channel623 having a flow direction 623 a and confining members 622, 624, 627,and 628. Although the confining members 622, 624, 627, and 628 arepositioned to define a flow path 623, in other embodiments one or all ofthe confining members 622, 624, 627, and 628 may not be used. The flowdirection 623 a aligns generally with the x-direction of the Cartesiancoordinate system illustrated in FIG. 6A. In the embodiment shown, thespatial filter 626 is arranged at a distance from the confining member622. In other embodiments, the spatial filter 626 may be arranged withinthe flow channel 623, mounted to any confining members or positionedrelative to any of the confining members. A detector (not shown) may bepositioned in any appropriate location to sense time modulated lightpassing through the filter 626 that has a component along the detectionaxis, z.

In FIG. 6A, the spatial filter 626 is arranged in the x-y plane of theCartesian coordinate system. The spatial filter 626 has a plurality ofmask features 670 arranged in a pattern. In particular, the maskfeatures 670 have repeating periodic patterns in the x-direction(referred to herein as the longitudinal direction) and at leastpartially extend along the lateral direction of the mask 626.Additionally, as shown in FIG. 6C, each of the mask features 670 has alength of either L₁ or L₂ in the x-direction. The lengths L₁ and L₂remain constant as the mask features 670 extend laterally across a widthof the spatial filter 626. In some embodiments, the ratio of the lengthsL₁ to L₂ is between 9:2 and 10:1. However, the ratio of the lengths L₁to L₂ may vary depending upon design and other criteria including, forexample, object size, object velocity, and ease of signal processing.

FIG. 6B shows the spatial filter 626 arranged in relation to a lightsource 612, detection region 623 b of a flow path 623, and a detector630. Objects move in the detection region 623 b along a flow direction623 a and emanate light. A light source 612 provides illuminating light612 a that illuminates the objects in the detection region 623 b. FIG.6B shows two possible intensity profiles 600, 601 of illuminating light612 a. The intensity profile 601 has substantially uniform intensityacross the detection region 623 b. For example, intensity profile 601could be provided by the sun or another wide area source. The intensityprofile 600 has an approximately Gaussian distribution. For example,intensity profile 600 could be provided by small area source or pointsource. Profiles 600 and 601 are but two possible profiles and invarious embodiments the profile of the illuminating light may vary fromthe uniform and Gaussian profiles illustrated. When the illuminatinglight is non-uniform, the shape of the profile of the illuminating light612 a is imposed on emanating light emanating from the objects and onthe time varying output signal 631.

FIG. 6C is a plan view of an enlarged portion of the spatial filter 626of FIG. 6A illustrating the mask features 670 in greater detail. Maskfeatures 670 include first mask features 670 a, 672 a that have firsttransmission characteristics, and second mask features 670 b, 672 b thathave second transmission characteristics different from the firsttransmission characteristics. For example, features 670 a, 672 a may bemore transmissive for a particular wavelength pass band than features670 b, 672 b. In the illustrated example, the more transmissive features670 a have lengths L₁ in the x-direction while the more transmissivefeatures 672 a have lengths L₂ in the x-direction. In the embodimentshown, the less-transmissive features 670 b have lengths L₁ in thex-direction while the less-transmissive features 672 b have lengths L₂in the x-direction.

In FIG. 6C, the mask features 670 alternate between first and secondsets of features 674 a, 674 b in the x-direction. The first and secondsets of features 674 a, 674 b provide for two different frequencycomponents in the time modulated light (and the time varying electricalsignal of the detector) as will be discussed subsequently. The first setof features 674 a is comprised of two of the transmissive regions 670 aarranged to either side of a single one of the less-transmissive regions672 b. The second set of features 674 b is comprised of two of theless-transmissive regions 670 b arranged to either side of a single oneof the transmissive regions 672 a.

FIG. 6D is a simplified plot 700 of the electrical signals generated inresponse to the modulated light that has passed through the maskfeatures 670 of the spatial filter 626 of FIGS. 6A and 6C. Thesimplified plot 700 presumes that the illuminating light has a uniformprofile and illustrates that the configuration of the mask features 670produces first and second signal components 702 and 704 that aresuperimposed in the resulting time domain electrical signal 706.

In FIG. 6D, first and second signal components 702 and 704 may both havea sinusoidal pattern. Signal components 702 and 704 represent amathematical decomposition of electrical signal 706 which is generatedin response to the modulated light that has passed through the maskfeatures 670 of the spatial filter 626 of FIGS. 6A and 6C. The firstsinusoidal signal component 702 is associated with the transmissiveregions 670 a (see, FIG. 6C) and the corresponding less-transmissiveregions 670 b (FIG. 6C) while the second signal component 704 isassociated with the transmissive regions 672 a (FIG. 6C) and thecorresponding less-transmissive regions 672 b (FIG. 6C).

The electrical signal 706 is the sum of the first signal component 702having a first frequency and the second signal component 704 having asecond frequency and is the result of alternating the first set offeatures 674 a (FIG. 6C) with the second set of features 674 b (FIG.6C). In the embodiment shown in FIG. 6D, the first frequency differsfrom the second frequency. In various embodiments, the second frequencymay be a multiple of the first frequency, e.g., three times the firstfrequency as shown in this example. In some embodiments, the analyzermay be configured to determine the trajectory depth in the flow path 623of the object 605 by analyzing the morphology of the signal 706 in thetime domain. In some embodiments, the analyzer may be configured todetermine the trajectory depth in the flow path 623 of the object 605 bytransforming the signal 706 from the time domain to the frequency domainand analyzing the transformed signal in the frequency domain.

FIGS. 6E and 6F show, respectively, a top view and a side view of thefilter 626 design of FIGS. 6A and 6C. Spatial filter 626 is used withilluminating light having an approximately Gaussian profile in anexemplary application to determine the trajectory depths in the flowpath 623 (possibly along with other characteristics) of objects 605 aand 605 b. In FIGS. 6E and 6F, the objects 605 a and 605 b are disposedwithin a flow channel 623 and are moving in a flow direction 623 a underthe filter 626. In this embodiment, the detector (not shown) would bepositioned above the filter 626 to sense light emanating from theobjects that has interacted with the filter 626. Thus, the objects 605 aand 605 b travel below the features 670 comprised of the first set offeatures 674 a and the second set of features 674 b.

FIG. 6G shows plots 700 a of the time-varying electrical signals 706 aand 706 b generated in response to the modulated light sensed from eachof the objects 605 a and 605 b moving relative to the spatial filter 626at different depths in the flow channel 623. Object 605 a is movingalong the flow channel 623 at a shallower depth (referenced from theposition of the spatial filter) in the flow channel 623 than object 605b, closer to the spatial filter 626 than object 605 b. Object 605 b ismoving along the flow channel 623 at a greater depth in the flow channel623 than object 605 a, farther from the spatial filter 626 than object605 a.

Signal 706 a corresponds to sensed light from object 605 a which isdisposed at a shallower depth within the flow channel 623 than object605 b relative to filter 626 and detector (not shown). Signal 706 bcorresponds to sensed light object 605 b which is disposed at a greaterdepth within the flow channel 623 than object 605 a relative to filter626 and detector (not shown).

As shown in the enlargement of the signal 706 a in FIG. 6G, the signal706 a includes “dips” in the peak region and “humps” in the troughregion. Similarly, the signal 706 b includes “dips” in the peak regionand “humps” in the trough region. In this particular embodiment, peakand valley regions correspond to the first frequency component and thedips and humps correspond to the second frequency component. The dipsare caused by the less-transmissive regions 672 b within the first setof mask features 674 a and the humps are caused by the more transmissiveregions 672 a within the second set of mask features 674 b.

The signal 706 a has an intensity profile with shorter transition timesbetween peak regions and trough regions and is more sensitive to themore transmissive regions 672 a and the less-transmissive regions 672 bsince more emanating light from object 605 a is transmitted or blockedby the regions 672 a and 672 b when compared with emanating light fromobject 605 b. This phenomenon results in a larger dip in the middle ofthe peak regions and larger humps in the middle of trough regions of thesignal 706 a relative to the dips and humps of signal 706 b.

For each signal 706 a and 706 b, the analyzer may be configured todetermine the trajectory depths in the flow channel 623 of the objects605 a, 605 b along the detection axis, z, by analyzing the signals 706a, 706 b in the time domain. In such an analysis, the analyzer circuitrymay be configured to determine the amplitudes of the dips and/or humpsand/or to determine the amplitude of the peak regions and the troughregions. In some embodiments, the analyzer is configured to compare theamplitudes of the dips and/or humps to the amplitude of the peak regionand/or trough region to determine the trajectory depth in the flowchannel 623 of the object 605 a and 605 b. This determination isinformed by mask features 670, which have a known size and pattern.Thus, the output signals 706 a and/or 706 b may be dependent on theknown mask pattern to allow for extraction of desired informationincluding depth. The determination of the trajectory depth in the flowchannel may additionally be informed by, for example, comparingcharacteristics of the output signals 706 a and/or 706 b to referenceoutput signals with known object light intensity, velocity, object size,and/or the known trajectory depth in the flow channel of other referenceobjects.

For each signal 706 a and 706 b, the analyzer may be configured todetermine the trajectory depths in the flow channel of the objects 605a, 605 b along the detection axis, z, by converting the time varyingsignals 706 a, 706 b to the frequency domain, for example, by using aFourier transform or fast Fourier transform (FFT). The analyzer thendetermines the trajectory depths in the flow channel of the objectsthrough analysis of the transformed signals. FIG. 6H shows plots 800 ofthe amplitudes of the electrical signals 706 a and 706 b converted tothe frequency domain. Plot 802 corresponds to signal 706 a for object605 a while plot 804 corresponds to the signal 706 b for object 605 b.Intensity peak I₂ corresponds to the amplitude of the peak and troughregions of the output signal and result from the more transmissiveregions 670 a (FIG. 6C) and the less-transmissive regions 670 b (FIG.6C) of the filter 626. Intensity peak I₃ corresponds to the amplitude ofthe humps and dips that result from the more transmissive regions 672 a(FIGS. 6C and 6E) and the less-transmissive regions 672 b (FIGS. 6C and6E) of the filter 626. As shown in plots 800, the intensity peak I₂ ofplot 802 is slightly larger than the intensity peak I₂ of plot 804 dueto the slightly larger peaks/valleys in signal 706 a. As shown in plots800, the intensity peak I₃ of plot 802 is much larger than the intensitypeak I₃ of plot 804 due to the much larger dips/humps in signal 706 a.The analyzer can be calibrated to determine trajectory depth in the flowchannel based on the amplitude of the intensity peaks I₂ and/or I₃and/or can be calibrated to determine trajectory depth in the flowchannel based on a ratio of I₂ and I₃. Utilizing the frequency domainanalysis including intensity peaks I₂ and/or I₃ can aid in thedetermination of the trajectory depth in the flow channel of the object.For example, analysis using the intensity peaks I₂ and I₃ allows fordirect comparison in the frequency domain of the amplitude of the peakand/or trough regions with the amplitude of the humps and/or dips.

Analysis of the trajectory depth in the flow channel of the object canbe performed using a ratio of the intensity peak I₂ relative to theintensity peak I₃ as illustrated in the plot 900 of FIG. 7. Plot 900illustrates I₂/I₃ intensity peaks measured on a logarithmic scaleplotted against velocity and trajectory depth in the flow channel. Datasets 902, 904, and 905 were generated from three separate sets ofexperiments with more than 2500 object events each are captured. Thesedata sets 902, 904, and 905 verify the robustness of the I₂/I₃ matrixmethodology. Parameters including fluidic flow rate and data acquisitionrate were altered in order to disturb the fluorescence intensityobtained, which is usually obtained and utilized as a matrix in flowcytometry. The analyte flow pump rate of 1.8 ill/min (micro-liters perminute) is sandwiched between two 30 μl/min sheath flows and dataacquisition rate is set to 400 kHz for the first data set 902. Thesecond data set 904 was performed with parameters of 4.5 μl/min analyte,75 μl/min sheath and data acquisition rate 600 kHz. The third data set906 was performed with parameters of 6 ill/min analyte, 100 μl/minsheath and data acquisition rate 600 kHz. The higher fluidic speed andhigher acquisition rate lead to a smaller intensity value. Although bothI₂ and I₃ vary with data sets 902, 904, and 906, using the I₂/I₃ ratioproduces an almost identical ratio value range from the three data sets902, 904, and 906. By utilizing mask features that produce the dip andhump phenomenon, many disturbance and variations effect the signals willlikely be canceled out in the matrix of I₂/I₃. The data sets 902, 904,and 906 show that observation of the I₂/I₃ ratio in log scale is veryrelevant to the known laminar flow velocity profile along the channeldepth. This is mainly attributed to the fact the amplitude of intensityprofile on mask features decreases exponentially with distance away fromthe mask features within the flow channel. Thus, the I₂/I₃ ratio can bea powerful metric to measure the depth of the object within the flowchannel.

FIG. 8A is an illustration of a portion of a fluidic device 1020,spatial filter 1026, and detector 1030. The fluidic device 1020 includesa flow channel 1023 with objects 1005 a, 1005 b disposed therein atdiffering depths. The objects 1005 a, 1005 b have light 1007 a, 1007 b,respectively, emanating therefrom as they move along the flow channel1023 in a flow direction 1023 a. As shown in FIG. 8A, the emanatinglight 1007 a, 1007 b from each of the objects 1005 a, 1005 b interactswith the filter 1026 to provide cones of light α₁ and α₂ that emergefrom transmissive regions of the filter 1026 and fall on the detector1030. The size and angles of the cones of light α₁ and α₂ are related tothe trajectory depth of the objects 1005 a, 1005 b within the flowchannel 1023 among other factors. Thus, the electrical signal producedby the detector 1030 includes at least one characteristic indicative ofthe angle of the cones of light α₁ and α₂. An analyzer (not shown) maybe configured to determine the depth of the objects 1005 a, 1005 b basedon the signal characteristic indicative of the angle of the cones oflight α₁ and α₂.

For example, for an excited object emitting an ideal spherical wave offluorescence, the emanating light 1007 a, 1007 b would project aGaussian distribution of optical intensity profile on the plane offilter 1026 governed by the equation

${{u\left( {x,y} \right)} = {\sqrt{\frac{2}{\pi}}\frac{1}{w}^{- \frac{({x^{2} + y^{2}})}{w^{2}}}}},$

where u is the Gaussian transverse amplitude with unity power flow, w isthe spot size. However, in most cases only a portion of the emanatinglight 1007 comprising, for example, cones of light α₁ and α₂ would passthrough portions of the filter 1026 and other intervening components(e.g., bounding member 1024) and fall on the detector 1030. Anyintervening components between the objects 1005 a, 1005 b and detector1030 would cause refraction of the cones of light α₁ and α₂ at theinterface of the components. This refraction is governed by knownprinciples according to Snell's law. Thus, using Gaussian distributionof optical intensity profile, the known characteristics of the filter1026, the measured characteristics of the electrical signal, and Snell'slaw one can determine the angle of the cones of light α₁ and α₂.

FIG. 8B shows a plot of the signal 1100 including a time modulationenvelope 1102 that results from the passage of the object 1005 a (FIG.8A) with cones of light α₂ along the flow path 1023 past detector 1030.The modulation envelope 1102 is disposed between a lower modulationfunction 1104 b comprising the minimum peaks of the troughs of thesignal 1100 and an upper modulation function 1104 a comprising themaximum peaks of the peaks of the signal 1100. Similarly, FIG. 8C showsa plot of the signal 1200 including a modulation envelope 1202 thatresults from the passage of the object 1005 b (FIG. 8A) with cones oflight α₁ along the flow path 1023 past detector 1030. The modulationenvelope 1202 is disposed between a lower modulation function 1204 bcomprising the minimum peaks of the troughs of the signal 1200 and anupper modulation function 1204 a comprising the maximum peaks of thepeaks of the signal 1200.

The modulation envelopes 1102 and 1202 are the signals generated by thedetector 1030 of FIG. 8A in response to the sensed light that falls onthe detector 1030. The trajectory depth in the flow channel of theobjects 1005 a, 1005 b may be determined based on one or morecharacteristics of the modulation envelopes 1102 and 1202.

For example, in some embodiments, the trajectory depth in the flowchannel of the objects may be determined using the amplitudes of thepeaks and/or troughs of the signal and/or the amplitudes of the humpsand dips within the peaks and/or troughs as previously discussed.

In some embodiments, the one or more characteristics may be anamplitude, e.g., peak amplitude, of the lower modulation function of themodulation envelope generated by the light emanating from an object. Insome embodiments, the one or more characteristics used to determineobject depth may be a width, e.g., full width half maximum (FWHM) width,of the lower modulation function of the modulation envelope generated bythe light emanating from an object.

As illustrated by FIGS. 8B and 8C, the maximum amplitude of the lowermodulation function 1104 b, 1204 b of the modulation envelope may benon-zero in some cases as the result of emanating light 1007 a, 1007 bpassing through more than one transmissive region of the spatial filter1026 and reaching the detector even when the object is over a blockingmask feature. The amount of modulation which may be characterized by themaximum value of the lower modulation function 1104 b, 1204 b can becorrelated to the trajectory depth of the objects 1005 within the flowchannel 1023.

FIGS. 5 through 8 refer to approaches for determining a trajectory depthposition of an object in a flow path with reference the detection axis(also referred to as z-axis and depth axis). The trajectory depthposition may be determined based on the time varying signal output fromthe detector and/or a frequency transform of the detector output signal.It will be appreciated that the time varying signal can be used todetermine longitudinal position of the object with reference to thex-axis as well as its trajectory depth position along the z-axis. Forexample, the analyzer may be configured determine the longitudinalposition of the object by analyzing the number of peaks and troughs inthe signal, wherein the peak or trough number can be correlated to alongitudinal position. In some embodiments, the analyzer may determine,separately or additionally, the velocity of the object along the x-axisbased on the time varying signal and/or based on a frequency transformthereof.

In some embodiments, spatial filter may be configured so that the objectposition in three dimensions and/or trajectory in three dimensions canbe determined. The analyzer can include circuitry that analyzes thedetector output signal to determine one or more of the longitudinalposition of the object along the x-axis, the lateral position of theobject along the y-axis, and the trajectory depth position of the objectalong the z-axis. In embodiments discussed herein the z-axis correspondsto the axis of detection.

In some embodiments, the spatial filter used for three dimensionalposition determination may include multiple types of features whereinfirst features are used to determine position along a first axis andsecond features, different from the first features, are used todetermine position along a second axis. The first features may bedisposed in a first portion of the spatial filter and the secondfeatures may be disposed in a second portion of the spatial filter.Alternatively, the first and second features may alternate along thespatial filter in the longitudinal direction. For example, the firsttype of features may be used to determine x-axis position and y-axisposition and a second type of features may be used to determine x-axisposition and z-axis position.

In other embodiments, the spatial filter may have features, e.g., asingle type of features, used to determine the position of the object inthree dimensions. In a these embodiments, the same features that areused to determine lateral position can also be used to determinedtrajectory depth position and longitudinal position, for example.

Measuring position along a reference axis, e.g., the lateral or depthaxes, can be accomplished using mask features that have a characteristicthat changes along the axis when the changing characteristic of the maskfeatures produces a discernible change in the output signal of thedetector. The changing characteristic in the mask features produces achange in phase, frequency, duty cycle, or some other characteristicthat is discernible in the detector output signal.

Some examples discussed below include mask features that have at leastone edge disposed at an angle with respect to the reference axis alongwhich the object position is determined. The at least one edge is notparallel or perpendicular to the reference axis. For this type of maskfeature, the position of the mask feature changes along the referencemeasurement axis, e.g., the lateral axis. Detector output signalsproduced by these mask features exhibit phase differences based on theposition of the object along the reference measurement axis. Someexamples discussed below include mask features that change in frequencywith respect to the measurement reference axis. These mask featuresproduce detector output signals having frequencies that depend on theposition of the object along the measurement reference axis.

FIG. 9A is an example of a spatial filter 926 that can be used todetermine lateral, longitudinal, and trajectory depth positions of anobject in a flow path. In this example, the analyzer can make adetermination of lateral position of the object based on the phase ofthe output signal as discussed in more detail below. The analyzer canmake a determination of depth position based on techniques previouslydiscussed, such as by analyzing the modulation envelope, e.g., analyzingthe lower modulation function, of the detector output signal.

The spatial filter 926 includes features 970 that have two edges 970 athat are perpendicular to the lateral axis, y, and two edges 970 b thatare not parallel or perpendicular to the lateral axis, making an anglewith the lateral axis. The interaction of the light emanating fromobjects 915 a, 915 b with the edges 970 b of the mask features 970generates output signals 980 a, 980 b that include a discernible phasedifference with respect to lateral positions, y_(a), y_(b) of theobjects 915 a, 915 b.

FIG. 9A depicts a first object 915 a flowing generally along thelongitudinal axis with constant velocity at a lateral position y_(a),and a second object 915 b flowing generally along the longitudinal axiswith constant velocity at a lateral position y_(b). Light emanating fromobjects 915 a, 915 b interacts with mask features 971, 970 to produceoutput signals 980 a, 980 b. The output signals 980 a, 980 b shown inFIG. 9A are idealized, and for simplicity ignore the rise and fall timesof the signal edges. The velocities of the objects 915 a, 915 b can bedetermined from the frequency components in the output signal producedby interaction of light emanating from the objects 915 a, 915 b with theperiodic mask features 970.

The light emanating from objects 915 a, 916 b interacts with a referencefeature 971, parallel to the lateral axis y, generating reference pulses981 a, 981 b at time t₀. Pulse 982 b is produced in the detector outputsignal 980 b at time t_(b) when light emanating from object 915 binteracts with mask feature 970. Pulse 982 a is produced in the detectoroutput signal 980 a at time t₀ when light emanating from object 915 ainteracts with mask feature 970. Pulse 982 a is shifted in time frompulse 982 b due to the lateral position of object 915 b relative to thelateral position of object 915 a. When the velocity of the objects isknown, the difference t_(b)−t_(a) can be used to determine the relativedifference in lateral position of the objects 915 a, 915 b. Thedifference t_(b)−t₀ can be used to determine an absolute lateralposition of object 915 b and the difference t_(a)−t₀ can be used todetermine an absolute lateral position of object 915 a.

The trajectory depth of objects 915 a, 915 b may be determined from theinteraction of light emanating from particles 915 a, 915 b with maskfeatures 970 based on the amount of offset of the troughs, as indicatedby the amplitude and/or peak of the lower modulation function of thedetector output signal, as discussed in connection with FIGS. 5-8. Notethat the objects 915 a, 915 b would not necessarily be flowing along theflow path at the same time as shown in FIG. 9A, but both signals 980 a,980 b are shown along the same time axis for comparison of theirrespective signals.

FIG. 9B illustrates another spatial filter 996 that can be used todetermine a three dimensional position of objects along the flow path,i.e., longitudinal position, lateral position, and trajectory depthposition. The spatial filter 996 includes mask features 997 useful fordetermining lateral position of objects in the flow path because themask features 997 have at least one edge that is not parallel orperpendicular to the lateral axis. These edges produce a phasedifference in the output signal due to the timing offset of the pulsesproduced by interaction between light emanating from objects and themask features as previously discussed. The mask features 997 are alsouseful for determining trajectory depth position of objects, e.g., byanalyzing the signal characteristics (peaks, troughs, dips and/or humps)in time and/or frequency domains as previously discussed in connectionwith FIGS. 5-8. For example, the trajectory depth position of objects inthe flow channel can be determined based on the amount of offset of thetroughs (i.e., the lower modulation function) of the detector outputsignal, as previously discussed in connection with FIGS. 5-8.

FIG. 10 illustrates another type of spatial filter 1056 having maskfeatures 1071, 1072, 1073 that change in frequency with respect to thelateral axis. Features 1071 are disposed along the longitudinal axis ofthe spatial filter at a first frequency, f₁, and at first constantlateral position, y₁. Features 1072 are disposed along the longitudinalaxis of the spatial filter at a second frequency, f₂, and at secondconstant lateral position, y₂. Features 1073 are disposed along thelongitudinal axis of the spatial filter at a third frequency, f₃, and atthird constant lateral position, y₃, where f₁>f₂>f₃, and y₁>y₂>y₃.

The lower portion of FIG. 10 depicts detector output signals 1081, 1083that are generated as light emanating from objects 1055 a, 1055 binteracts with mask features 1071, 1073. As shown in FIG. 10, thefrequency of signal 1081 is greater than that of 1083. The frequency ofthe detector output signal can be used to determine lateral position ofthe objects. For example, signals 1081 having frequency f₁ are generatedby objects 1055 a flowing at a lateral position in the region of maskfeatures 1071, e.g., y₁±½ the width of the features 1071, where featurewidth is measured along the y axis. Signals 1082 having frequency f₂ aregenerated by objects 1055 b flowing at a lateral position in the regionof mask features 1072, e.g., y₂±½ the width of the features 1072,wherein feature width is measured along the y axis. Thus, the frequencyof the detector output signal can be used to determine lateral positionof the object. Note that the objects 1055 a, 1055 b would notnecessarily be flowing along the flow path at the same time as shown inFIG. 10, but both signals 1081, 1083 are shown along the same time axisfor comparison of their respective signals.

Additionally, the trajectory depth in the flow channel of the object canbe determined from the interaction of light emanating from particles1055 a, 1055 b with mask features 1071, 1072, 1073 based on the amountof offset of the troughs (i.e., the lower modulation function) of thedetector output signal, as previously discussed in connection with FIGS.5-8. Furthermore, the longitudinal position of the objects can bedetermined based on the pulse number in the output signal. For example,considering object 1055 a, the first output pulse, P₁, at frequency f₁is associated with longitudinal position x₁, the second output pulse,P₂, at frequency f₂ is associated with longitudinal position x₂, and soforth. Thus, detector output signals generated by the interaction oflight emanating from objects 1055 a, 1055 b interacting with spatialfilter 1056 can be used to determine position of the objects in threedimensions. For the embodiments shown in FIGS. 9-10, the depth axis isalong the detection axis of the detector.

In some embodiments, two or more types of mask features may be used todetermine the position of an object in multiple dimensions, e.g., alonglongitudinal, lateral, and trajectory depth axes. FIG. 11A illustrates aspatial filter 1120 that includes first and second regions 1140, 1150,wherein a first group of spatial features useful for determining depthposition are arranged placed within the first region 1140 and group ofspatial features useful for determining lateral position are arrangedplaced within the second region 1150. In some embodiments, the firstgroup of features is useful for determining lateral position and thesecond group of features is useful for determining depth position. Insome embodiments, as illustrated in FIG. 11B, the spatial filter 1121includes more than two regions, wherein different groups of spatialfeatures may be placed in the regions. For example, the first group ofspatial features used to determine the trajectory depth position may beplaced in regions 1141 and 1142 and the second group of spatial featuresused to determine lateral position can be disposed in regions 1143 and1144. The features in all regions 1140, 1150, 1141, 1142, 1143, 1144 maybe used to determine longitudinal position in some embodiments.

FIGS. 12-15 illustrate spatial filters that are useful for determininglateral position of an object in the flow path. These features usefulfor determining lateral position may be used in conjunction withadditional features (not shown in FIGS. 12-15) of the spatial filter todetermine trajectory depth position, as discussed in connection with thespatial filters of FIGS. 11A and 11B.

FIG. 12 shows a perspective view of a portion of a fluidic device 1320and an embodiment of a spatial filter 1326. The fluidic device 1320includes a flow path 1323 having a flow direction 1323 c, and confiningmembers 1324, 1327, and 1328. The confining members 1324, 1327, and 1328are positioned to define the flow path 1323. The flow direction 1323 caligns generally with the x-direction of the Cartesian coordinate systemillustrated in FIG. 12. In the embodiment shown, the spatial filter 1326is mounted along a confining member (not shown) that extends generallyalong the x-y plane. In other embodiments, the spatial filter 1326 maybe disposed externally to or within the flow channel 1323, and/orpositioned relative to any of the other illustrated confining members1324, 1327, and 1328. A detector may be positioned in any appropriatelocation to sense modulated light passing through the filter 1326. Thedetector is positioned so that it is capable of detecting light having acomponent that lies along the detection axis (i.e., the z-axis). In theillustrated embodiments, the detection axis is selected to be the depthaxis.

For example, for a fluidic device 1320 and spatial filter 1326 havingthe orientation of FIG. 12, an excitation light source (also not shownin FIG. 12) can be oriented below confining member 1324 and a detector(not shown in FIG. 12) can be oriented above the filter 1326. In such anarrangement, excitation light from the light source passes throughconfining member 1324 and optically interacts with objects travelingwithin a detection region of the flow path 1323. The excitation lightcauses the objects to emanate light in all directions and a portion ofthe emanating light from the objects is spatially modulated by filter1326. A detector positioned above the spatial filter senses thespatially modulated light and, in response, generates a time varyingsignal.

In FIG. 12, the spatial filter 1326 is arranged in the x-y plane of theCartesian coordinate system. The spatial filter 1326 includes maskfeatures that allow for a determination of a lateral position (i.e., aposition in the y-direction of the Cartesian coordinate system) of anobject within the flow path 1323. The spatial filter 1326 may alsoinclude mask features (not shown) that allow for determination of atrajectory depth position (i.e., a position in the z-direction of theCartesian coordinate system) of an object within the flow path 1323.Each mask feature may be used to determine longitudinal position (i.e.,a position in the x-direction of the Cartesian coordinate system) of anobject within the flow path 1323.

FIG. 13A shows a top plan view of the spatial filter 1326 of FIG. 12.The spatial filter 1326 has mask features 1370 that each have a length Lwith respect to a flow direction 1323 c of the flow channel 1323, of alength that changes as each mask feature 1370 extends across the lateralaxis y of the flow channel 1323. The mask features 1370 includetransmissive and less transmissive regions and edges of sometransmissive regions extend at a non-perpendicular angle with respect tothe flow direction 1323 c of the flow channel 1323. Although embodimentsillustrated herein show triangular mask features, the mask featurescould alternatively be truncated triangles, parallelograms, or any othershape that has an edge that varies non-perpendicularly with respect tothe flow direction. FIG. 13A additionally shows objects 1305 withdifferent trajectories of flow across the filter 1326 as connoted bypaths I-III. As shown in FIG. 13A, the mask features 1370 are periodicwith respect to the flow direction 1323 c of the flow channel 1323 andduty cycle of the mask features 1370 changes along the lateral y-axis yof the flow channel 1323.

FIG. 13B is a plot 1500 that shows detector output signals 1502, 1504,and 1506 that result from the objects 1305 flowing across the filter1326 along paths I-III. In particular, signal 1502 corresponds with pathI, signal 1504 corresponds with path II, and signal 1506 correspondswith path III. As shown plot 1500, the characteristics of the signals1502, 1504, and 1506 are correlated to the geometry of the mask features1370 and correspond to the lateral location of the object 1305 (FIG.13A) within the flow channel 1323. Thus, the signal 1502 of path I has arelatively small duration of non-zero amplitude due to the small lengthL of the transmissive regions 1370 along path I. The configuration offilter 1326 also allows for determination of the trajectory of theobjects 1305 in the x-y plane based upon changes in the characteristicsof the signals. For example, signal 1504 experiences an increasedduration of the signal in a peak region as the object 1305 flows alongthe filter 1326 in the x and y directions. This increase results from agradually increasing length L (in the x direction) of the transmissiveregions 1370 along path II, in addition to a small increase from thelateral shift (in y direction).

FIG. 14 shows a perspective view of a portion of a fluidic device 1620and an embodiment of a spatial filter 1626. The fluidic device 1620includes a flow path 1623 having a flow direction 1623 c, and confiningmembers 1624, 1627, and 1628. The confining members 1624, 1627, and 1628are shown in the illustrated embodiment as being positioned to definethe flow path 1623, but in other embodiments the confining members maynot be present. The flow direction 1623 c aligns generally with thex-direction of the Cartesian coordinate system illustrated in FIG. 14.In the embodiment shown, the spatial filter 1626 is mounted along aconfining member (not shown) that extends generally along the x-y plane.In other embodiments, the spatial filter 1626 may be disposed externallyto or within the flow channel 1623, and/or positioned relative to any ofthe illustrated confining members 1624, 1627, and 1628. A detector ormultiple detectors (not shown) may be positioned in any appropriatelocation to sense modulated light passing through the filter 1626. InFIG. 14, the spatial filter 1626 has mask features that allow for adetermination of a lateral position (i.e., a position in the y-directionof the Cartesian coordinate system) of an object within the flow channel1623.

In some embodiments, the spatial filter may include the mask features asshown in spatial filter 1626 and may also include other mask features(not shown) that allow for determination of a trajectory depth position(i.e., a position in the z-direction of the Cartesian coordinate system)of an object within the flow path 1623. Each mask feature may be used todetermine longitudinal position (i.e., a position in the x-direction ofthe Cartesian coordinate system) of an object within the flow path 1623.

FIG. 15A shows a side plan view of the spatial filter 1626 of FIG. 14.The spatial filter 1626 has mask features 1770 with two alternatingsizes and orientations. In the exemplary embodiment, first transmissivemask features 1784 are separated from second transmissive mask features1780 by less-transmissive mask features 1782 in a pattern ofinterdigitated triangles. In one embodiment, the first mask features1784 comprise first triangles, e.g., isosceles triangles, with a firstangle and second mask features 1780 comprise second triangles, e.g.,isosceles triangles, with a second angle that differs from the firstangle. The first mask features 1784 have a length L₁ with respect to thelongitudinal x-axis that changes as each first mask feature 1784 extendsalong the lateral y axis of the flow channel 1723. Additionally, thesecond mask features 1780 have a length L₂ with respect to thelongitudinal x-axis that changes as each second mask feature 1780extends along the lateral y axis. FIG. 15A additionally shows objects1305 a, 1305 b traveling at two different trajectories of flow acrossthe filter 1626 at differing lateral positions 1306 a, 1306 b.

FIG. 15B is a plot 1800 that shows detector output signals 1802 and 1804that result from objects flowing across the filter 1626 along paths(PATH 1, PATH II) at different lateral positions. In particular, signal1802 corresponds to an object traveling along PATH I where L₁≈L₂ forfirst transmissive mask feature 1784 and second transmissive maskfeatures 1780. Signal 1804 corresponds to an object traveling along PATHII where L₁>L₂. As shown in plot 1800, the characteristics of thesignals 1802 and 1804 are correlated to the geometry of the maskfeatures 1770 and correspond to the lateral position of the objects. Forexample, the detector output signal generated for an object travelingalong a trajectory where L₁≈L₂ has a single predominant frequencycomponent. The detector output signal generated for an object travelingalong a trajectory where L₁ is different from L₂ has two predominantfrequency components. Thus, the lateral position of the object can bedetermined using a Fourier transform to convert the signal to thefrequency domain and then analyzing the intensity peaks of thetransformed signal to determine the predominant frequency components.These frequency components map to a lateral position of the flow path.The configuration of filter 1726 also allows for determination of theposition and/or trajectory of the objects in the x-y plane based uponchanges in the characteristics of the signals.

Spatial masks that are useful for determining lateral position ofobjects are illustrated in FIGS. 12-15 as having patterns of triangularfeatures. It will be appreciated mask features other than triangularfeatures can be used in various embodiments. Using spatial filters tomodulate light as described herein, the position of an object in a flowpath can be determined using a spatial filter that has mask featureswith a changing characteristic such as an edge between first and secondmask features having a non-perpendicular and non-parallel orientationwith respect to the flow direction along the trajectory depth directionof the flow channel. The changing characteristic causes a change in atleast one of the duty cycle, frequency, or phase in the time-varyingsignal generated by the detector. In many applications it may be usefulto determine the velocity of the objects. Object velocity can bedetermined by determining the frequency of the transitions in the timevarying output signal and/or by transforming the time varying signal toa frequency domain signal and analyzing the dominant frequencies havingthe largest amplitude.

Some systems are capable of determining the position of an object inthree dimensions. In some embodiments, systems that determine a threedimensional position of objects use two (or more) masks oriented indifferent planes. For example a first mask that includes a first groupof mask features may be used for detecting lateral position and a secondmask includes a second group of mask features may be used for detectingdepth position. In such a system, a first detector is arranged relativeto the first mask so that the first detector detects light emanatingfrom objects flowing along a flow path that is spatially modulated bythe first mask. The first detector generates a first output signal inresponse to the emanating light that is spatially modulated by the firstmask. A second detector is oriented relative to the second mask so thatthe second detector detects light emanating from objects flowing along aflow path that is spatially modulated by the second mask.

FIG. 16 shows a perspective view of a portion of a fluidic device 1820and two spatial filters. A first spatial filter 1826 is oriented withrespect to the flow path 1823 extending generally in the x-y plane and asecond spatial filter 1827 is oriented with respect to a flow path 1823extending generally in the x-z plane. The flow direction 1823 c alignsgenerally with the longitudinal x-direction of the Cartesian coordinatesystem illustrated in FIG. 16.

The spatial filter 1826 has a first group of mask features that allowfor a determination of a depth position (i.e., a position in thez-direction of the Cartesian coordinate system) of an object within theflow channel 1823. The spatial filter 1827 includes a second group ofmask features that allow for a determination of a lateral position(i.e., a position in the y-direction of the Cartesian coordinate system)of an object within the flow channel 1823. Either or both groups of maskfeatures may be used to determine longitudinal position (i.e., aposition in the x-direction of the Cartesian coordinate system) of anobject within the flow channel 1823.

FIG. 17 shows a flow diagram of a method of analyzing a sample. As partof an initialization 2710 for the system, objects of a known size and/orluminescence are passed through a flow path relative to a spatial filterat different depths and/or lateral positions so that the system can becalibrated. The primary purpose of the calibration step is to perform asystem validation to verify that the system is tuned properly, to ensurethat all the parameters are set properly, and that the system cancorrectly identify the type of objects and successfully determine theirposition, velocity and depth trajectory as the case may be. For example,prior to running the actual sample to be measured, and subsequently infrequent time intervals thereafter, a pre-made mixture of certain objectsizes is applied through the flow channel to verify that the system cancorrectly determine their position, velocity and trajectory depth in theflow channel. The objects may additionally be coated with differentmaterials to simulate for example different intensity levels and/orother system aspects. If the system is found to drift over time awayfrom the known object dimensions, the system parameters can be tuned tocompensate and bring it back to within accuracy specification. Thecalibration step is optional. The system information may also begathered over time during regular use of the system by applying amachine learning algorithm.

Light from the sample containing an object of interest is sensed 2720 asthe object moves through the flow path relative to the spatial filter.As discussed previously, the sensed light is modulated according to maskfeatures. An electrical output signal is generated 2730 in response tothe sensed light. The signal is analyzed 2740 to determine at least atrajectory depth of the object within the flow path. Additional steps2750 and/or alternative steps can be performed as desired to support themethod described.

Although the subject matter has been described in language specific tostructural features and/or methodological acts, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the specific features or acts described above.Rather, the specific features and acts described above are disclosed asrepresentative forms of implementing the claims.

What is claimed is:
 1. An assembly, comprising: at least one spatialfilter having a length along a longitudinal axis of a flow path and awidth along a lateral axis of the flow path, the spatial filter havingmask features disposed at least partially along the length of thespatial filter and extending at least partially across the width of thespatial filter, the mask features including at least first mask featureshaving a first transmission characteristic and second mask featureshaving a second transmission characteristic different from the firsttransmission characteristic; at least one detector positioned to detectlight with respect to a detection axis, the detection axis making anon-zero angle with respect to the longitudinal and lateral axes, thedetected light emanating from at least one object and modulatedaccording to the mask features as the object moves along thelongitudinal axis, the detector configured to generate a time-varyingelectrical signal in response to the detected light, the time-varyingelectrical signal including information about a depth of the object inthe flow path along the detection axis; and an analyzer configured todetermine the depth of the object based on the signal.
 2. The assemblyof claim 1, wherein: the spatial filter is positioned between a lightsource that provides measurement light and the object; and the spatialfilter interacts with the measurement light to provide spatiallymodulated measurement light.
 3. The assembly of claim 1, wherein: thespatial filter is positioned between the object and the detector; andthe light emanating from the object interacts with the spatial filter toprovide modulation of the sensed light.
 4. The assembly of claim 1,wherein: the light emanating from the object interacts with the spatialfilter to provide cones of light that emerge from the mask, the angle ofeach cone related to the depth of the object; and the time-varyingsignal includes at least one characteristic associated with the angle ofthe cone.
 5. The assembly of claim 1, wherein: the mask features includea first set of features alternating with a second set of features; thefirst set of features comprises at least two transmissive regions havinglength L1, each transmissive region of length L1 separated from anothertransmissive region of length L1 by a less-transmissive region of lengthL2, where L1>L2; and the second set of features comprises at least twoless-transmissive regions having length L1, each less-transmissiveregion of length L1 separated from another less transmissive region oflength L1 by a transmissive region of length L2.
 6. The assembly ofclaim 1, wherein: the time-varying signal generated in response to themodulated light includes a first signal component of a first frequencysuperimposed with a second signal component of a second frequency, thefirst frequency being different from the second frequency.
 7. Theassembly of claim 6, wherein the second frequency is a multiple of thefirst frequency.
 8. The assembly of claim 6, wherein the analyzer isconfigured to determine the depth of the object in the flow channel bycomparing the amplitude of the first signal component to the amplitudeof the second signal component.
 9. The assembly of claim 1, wherein: thetime-varying signal generated in response to the modulated lightincludes a first signal component of a first frequency superimposed witha second signal component of a second frequency, the first frequencybeing different from the second frequency; and the analyzer isconfigured to perform a Fourier transform of the time-varying signal andto compare an amplitude of the Fourier transform signal at the firstfrequency to an amplitude of the Fourier transform signal at the secondfrequency to determine the depth position of the object.
 10. Theassembly of claim 9, wherein the analyzer is configured to determine thedepth of the object in the flow channel based on a difference betweenthe amplitude of the Fourier transform signal at the first frequency andthe amplitude of the Fourier transform signal at the second signalcomponent.
 11. The assembly of claim 1, wherein: the signal generated inresponse to the sensed light has a modulation envelope, the modulationenvelop defined by a lower envelope function and an upper envelopefunction; and the analyzer is configured to determine the depth of theobject based on one or more characteristics of the modulation envelope.12. The assembly of claim 11, wherein the depth is determined based onan amount of modulation of the lower envelope function.
 13. The assemblyof claim 1, wherein: an edge between the first and second mask featuresfor at least some of the mask features extends at an angle that is notperpendicular and not parallel to the longitudinal axis of the mask; andthe spatial filter includes at least one reference feature that issubstantially parallel to the lateral axis.
 14. The assembly of claim13, wherein the time-varying signal also includes information about aposition of the object along at least one of the lateral andlongitudinal axes.
 15. The assembly of claim 1, wherein: the maskfeatures include a first set of features alternating with a second setof features; the first set of features comprises at least twotransmissive regions having length L1, each transmissive region oflength L1 separated from another transmissive region of length L1 by aless-transmissive region of length L2, where L1>L2; and the second setof features comprises at least two less-transmissive regions havinglength L1, each less-transmissive region of length L1 separated fromanother less transmissive region of length L1 by a transmissive regionof length L2, wherein each mask feature has at least one edge that isnot parallel and not perpendicular to the longitudinal axis of the mask.16. The assembly of claim 1, wherein the mask features have a changingfrequency across the lateral axis of the spatial filter.
 17. Theassembly of claim 1, wherein the analyzer is further configured todetermine position of the object along the lateral and longitudinal axesbased on the signal.
 18. The assembly of claim 1, wherein: the spatialfilter includes: a first group of mask features; and second group ofmask features; the detector is configured to generate a time varyingelectrical signal, the time varying electrical signal having a firstportion generated in response to detected light modulated according tothe first group of mask features and having a second portion generatedin response to detected light modulated according to the second group ofmask features.
 19. The assembly of claim 18, wherein at least one edgebetween a first mask feature and a second mask feature of the firstgroup of mask features is not parallel or perpendicular to thelongitudinal axis.
 20. The assembly of claim 19, wherein the first groupof mask features includes one or more triangular mask features.
 21. Anassembly, comprising: a first spatial filter arranged along an x-y planeof a flow path characterized in three dimensional space by x, y, and zaxes, the first mask having a first group of mask features; a secondspatial filter arranged along an x-z plane of the flow path, the secondmask having a second group of mask features; a first detector positionedto detect light emanating from at least one object and modulatedaccording to the first group of mask features as the object moves alongthe flow path, the first detector configured to generate a firsttime-varying electrical signal in response to the detected lightmodulated according to the first group of mask features; a seconddetector positioned to detect light emanating from at least one objectand modulated according to the second group of mask features as theobject moves along the flow path, the second detector configured togenerate a second time-varying electrical signal in response to thedetected light modulated according to the second group of mask features;and an analyzer configured to determine a position of the object alongthe x, y, and z axes based on the first and second time-varying signals.22. The assembly of claim 21, wherein at least one edge between a firstmask feature and a second mask feature of the first group of maskfeatures is not parallel or perpendicular to a flow direction of theflow path.
 23. A method, comprising: detecting light emanating fromobjects moving along a flow path, the emanating light modulated by atleast one spatial filter having a length along a longitudinal axis of aflow path and a width along a lateral axis of the flow path, the spatialfilter having mask features configured to modulate light, the detectedlight having a component along a detection axis that makes a non-zeroangle with respect to the longitudinal and lateral axes; generating anelectrical output signal in response to the detected light; anddetermining a depth of the object in the flow path along the detectionaxis based on the output signal.
 24. The method of claim 23, whereindetermining the depth of the object comprises performing a time domainanalysis.
 25. The method of claim 23, wherein determining the depth ofthe object comprises performing a frequency domain analysis.